Summary This proposal is for a COVID-19-related Administrative Supplement for RO1HL142111, ?Ubiquitin regulation of K Channels in Health and Disease?. We propose to adapt/apply tools and approaches including, nanobody development and optical assays of membrane proteins, that we have developed for studies in the parent proposal to develop novel solutions for COVID-19. There is an ongoing global pandemic in which a novel Coronavirus, SARS-CoV-2, causes Coronavirus Disease-2019 (COVID-19) that is lethal to a subset of the infected population. To date, there are >1.5 million confirmed COVID-19 cases worldwide with >70,000 fatalities. In the United States, best case scenario projections indicate 100,000 ? 240,000 lives will be lost to COVID-19, with a worst case scenario of >2 million deaths. The current pandemic follows two other regional outbreaks of coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV) in 2002, and Middle Eastern respiratory syndrome (MERS-CoV) in 2012, that caused epidemics with fatality rates of 10% and 36%, respectively. All three coronaviruses represent examples of animal to human transmission of infection. Given a high zoonotic reservoir of novel coronaviruses and persistence of close animal/human contact in various parts of the world, the chances of future viral epidemics/pandemics beyond the current crisis is high. Hence, fatal diseases caused by viral infections represent current and future widespread public health challenges, highlighting an urgent need for general strategies that can combat not only COVID-19 but also other future viral outbreaks. SARS-CoV-2 is an enveloped virus that gains access to host cells by using a receptor binding domain (RBD) on a viral surface spike protein (S) to bind to membrane-bound angiotensin-converting enzyme 2 (ACE2) receptors on target cells. Preventing the SARS-CoV-2 S protein/ACE2 receptor interaction is the main principle behind the beneficial effects of neutralizing antibodies in recovered patients, and the scientific premise for an ongoing clinical trial for administration of soluble ACE2 ectodomain. There are ongoing efforts to develop neutralizing monoclonal antibodies (mAb) against SARS-CoV-2 S protein that disrupt interaction with ACE2. While potentially effective, limitations of this approach include high cost, time-consuming, incompatibility with inhalation formulations, and potential for viral mutations that result in escape from mAb-mediated neutralization. This proposal is based on the hypothesis that engineered single-domain antibodies (nanobodies) with high avidity for SARS-CoV-2 S protein can be generated to overcome some of the limitations of neutralizing mAbs and provide potential therapeutic leads for COVID-19. We propose three aims: 1) Isolate and characterize neutralizing and non- neutralizing nanobodies against SARS-CoV-2 S protein; 2) Develop cell-based high throughput fluorescence assay to probe SARS-CoV-2 S protein/ACE2 interaction and internalization; 3) Comparatively evaluate the capacity of engineered nanobodies to disrupt SARS-CoV-2 S protein/ACE2 interaction and internalization